The long-term goal of this proposal is to understand the cellular and molecular mechanisms that control cell fate determination in the developing vertebrate nervous system, with a particular emphasis on the choice between neuronal and glial fates. Specifically, we propose to study the function and regulation of Olig1 and Olig2, two basic helix-loop-helix (bHLH) transcription factor genes we have recently isolated that are specifically expressed in the oligodendrocyte lineage (Zhou et al. (2000) Neuron 25:331). In Specific Aim I of this proposal, we will determine whether the Olig genes are essential determinants of the oligodendrocyte fate in vivo, by generating Olig1 and 2-deficient mice and analyzing the phenotypes of various allelic combinations of these mutations, using an extensive battery of molecular markers. In Specific Aim II, we will determine whether expression of Olig genes marks commitment to the oligodendrocyte lineage, by using vital markers incorporated into the gene targeting cassettes to isolate Olig1- and Olig2-expressing cells by flow cytometry. The developmental capacities of these isolated cell populations will be tested by in vivo transplantation and by in vitro cell culture. In the second part of this proposal we will investigate the regulation of Olig gene expression and its functional interactions with other determinants of neural cell fate in the spinal cord. The expression of Olig genes defines a restricted zone from which oligodendrocyte precursors will emerge several days later. In Specific Aim III, we will carefully map the relationship of the Olig gene expression domain to the domains of expression of other known regulators of spinal cord cell fate, using double- and triple-labeling with antibodies and/or cDNA probes and laser scanning confocal fluorescence microscopy. These latter regulators include components of a recently identified homeodomain code that specifies different progenitor domains (Briscoe et al. (2000) Cell 101:435); neurogenic bHLH factors and Notch ligands. In Aim IV, we will functionally test hypotheses for the regulation of Olig gene expression suggested by correlative data obtained in Aim III, using retrovirus- or electroporation-mediated gene transfer to mis-express candidate regulatory genes in the chick spinal cord and determine their effect on the domain of Olig gene expression, and vice-versa. An understanding of the cellular and molecular mechanisms that control oligodendrocyte lineage determination in neural progenitor cells is an essential prerequisite to the controlled manipulation of such cells for transplantation therapy of neurological diseases such as Multiple Sclerosis.